The present invention relates to a method and apparatus for measuring the pH of a cell culture solution.
In order to grow and proliferate cells, the pH of a culture solution containing the cells must be within a range suitable for proliferation. During preparation or storage of such a cell culture solution, however, carbon dioxide which is contained in the cell culture solution is released, and the pH is increased, so that the pH is often deviated from the proliferation suitable range.
Therefore, the pH is measured by a method in which, for example, the color change of phenol red that is usually contained in a cell culture solution is visually checked, or that in which the measurement is performed while pH electrodes are immersed in a cell culture solution. However, these methods have the following problems.
In the case where the color change of phenol red contained in a cell culture solution is visually checked, an erroneous check may be caused. By contrast, in the case where pH electrodes are immersed in a cell culture solution, when the pH electrodes are not sufficiently sterilized, contamination due to bacteria or the like may occur.
As a method of measuring the pH of a cell culture solution which is free from problems such as an error due to a visual check, and contamination in the case where pH electrodes are used, there is the following method (see JP-B-6-34754).
JP-B-6-34754 describes the followings.
A pH measuring method is provided which is a method of measuring the pH of a cell culture solution in which the pH is measured based on absorption of visible light in a cell culture solution that includes: a cell culture medium; serum; and an indicator having two or more kinds of absorption peaks in the wavelength region of visible light, wherein, based on a linear relationship between the pH and the logarithms of absorbances at two wavelengths of absorption peaks that are obtained by transmitting visible light through a cell culture solution in which the pH is known, the value of the pH is obtained from the value of the logarithm of a ratio of absorbances of absorption peaks that are measured in a cell culture solution specimen in which the pH is not known.
Furthermore, a pH measuring method is provided which is a method of measuring pH of a cell culture solution in which the pH is measured based on absorption of visible light in a cell culture solution that includes: a cell culture medium; serum; and an indicator having two or more kinds of absorption peaks in the wavelength region of visible light, wherein, based on a linear relationship between the pH and the logarithms of a ratio of differences between absorbances at two wavelengths of absorption peaks that are obtained by transmitting visible light through a cell culture solution in which the pH is known, and an absorbance at a wavelength where absorption peaks do not exist, the value of the pH is obtained from the value of the logarithm of a ratio of a difference of similar absorbances that are measured in a cell culture solution specimen in which the pH is not known.
The method of measuring the pH of a cell culture solution is characterized in that a cell culture solution that includes: a cell culture medium; serum such as fetal bovine serum; and an indicator, is poured into a transparent container, the cell culture solution is irradiated with visible light, and the pH is calculated from the transmission spectrum or reflection spectrum.
In a cell culture solution, usually, phenol red for detecting a change of the pH is contained at a low concentration which does not harm cells. In the visible light range, at such a low concentration, phenol red has peaks in the vicinities of 430 to 440 nm and 560 nm, and an isosbestic point at 480 nm. In a pH range of 6.8 to 7.6 where cells can grow, as the pH is further lowered, the absorption peak in the vicinity of 430 to 444 nm is more increased, and that in the vicinity of 560 nm is more decreased. When absorption due to only the phenol red is obtained, by taking a ratio of absorption in the vicinity of 430 to 440 nm to that in the vicinity of 560 nm, the plot shows one curve, and the pH of the culture solution can be calculated from a ratio of the two peaks.
The following is known as a reference example of a technique in which two parameters, i.e., the temperature and pH of the culture medium that are important for, in cell culture such as in artificially fertilized cell culture, regulating the environment to ensure healthy cell growth are monitored (see JP-T-2009-533053).
The embodiment in the reference example shown in FIG. 9 includes an incubator 202 having trays 204 upon which culture dishes 206 are carried. Other culture vessels such as flasks may be used. Moreover, the incubator may be of any size or construction. Each culture dish is accompanied by a pH sensor and a temperature sensor associated with a cuvette 208 of a medium. The sensors perform measurements of the pH and temperature of the medium in the cuvette and hence of the pH and temperature of the media in the culture dish without the need for the light sensors and thermocouple to be directed into the culture dish. Hereinafter, these units are referred to as “reader units”. In the embodiment, the reader units optically perform the pH measurement by using light emitting diodes (LEDs) as a light source, and the temperature measurement by using the thermocouple.
An embodiment of the reader units includes a fully sealed unit so that it can withstand spillages, with packaging made from a suitable plastic which can be cleaned and sterilized. In other cell culture applications using large dishes or flasks, it may be possible to immerse the unit in the actual solution being monitored. In this case, if phenol red is not dissolved in the solution, an optode with an immobilized indicator may be used. The reader unit may be either re-chargeable, or have a battery which either lasts a sufficiently long time, or is replaceable.
The reader unit 210 has a wireless communication capability with respect to a slave receiver/transmitter unit 212. The slave receiver/transmitter unit 212 is connected wirelessly to a data logger 214 which records the data from the reader units. The data logger 214 has a download capability with respect to a computer system 216 which displays and stores the details of the temperature and the pH. Alternatively, the slave receiver/transmitter unit 212 may be hard wired to the data logger 214.
The complete system is modular and expandable. A central data logger is a repository of data and can accommodate the data streams from multiple readers. The data are downloadable to a PC, and a suitable piece of software for downloading and presenting the data forms part of the system. Reader units can be used to monitor the conditions in an incubator and control feedback, but the use of a reader per culture vessel enables tracking of the history of the individual culture vessels. When the vessel is outside the incubator for inspection, medium changes, etc., it is most susceptible to variations in temperature and pH, so this is really the crucial time to monitor the situation. In such a situation, the reader unit 210a can transmit wirelessly directly to the data logger 214.
Since much of the culture cycle will be spent inside a metal clad incubator, it is envisaged that a slave receiver/transmitter can be placed inside or outside the incubator to receive the wireless signals from the units during these periods. This unit can be connected to a main logger unit situated outside the incubator and may be connected wirelessly or be hard wired. Alternatively, the reader unit may be hard wired to the data logger or the data logger may have antennae which are inserted into the incubator (thereby removing the need for slave receiver/transmitter units). Since the embodiment is one where there is a central data logger receiving data from multiple incubators (and multiple dishes therein), however, greater flexibility would be provided by having a slave receiver/transmitter unit with each incubator. If the incubators are clad in a material which transmits radio signals, the receiver/transmitter can also directly transmit to the logger unit.
When the culture dish 206a is outside the incubator 202, therefore, the reader unit 210a can transmit directly to the data logger 214.
In the case where the reader unit is being used to monitor the history of an individual vessel, it needs to stay associated with that vessel, and a holder can be used which holds both the vessel and reader unit so that they can be transported about together.
In the embodiment, the reader units wirelessly transmit data. Whilst, inside the incubator, the data will be received by the slave unit. The main logger unit also looks for the data stream, and does not receive it when the units are outside the incubator, the slave units will not receive the signals through the metal cladding of the incubator. Alternatively, the slave receiver/transmitter unit may be placed on the outside of the incubator with an antenna inside and outside the incubator so that it always receives the signal. In order to conserve power while the reader unit is in the incubator, the reader unit may not transmit data continuously, but at a pre-determined time interval. When the reader unit is once outside the incubator, the reader can perform transmission more frequently since this is the time when changes are likely to occur more rapidly. One way of causing the reader to know that it is outside the incubator is to use a photodiode and look for changes in ambient light. Inside the incubator, it will generally be dark. The reader units will also have warning indicators which, when the pH or the temperature starts to go outside of the acceptable range, warn that the vessel should be put back in the incubator. If the cycle is complete and/or the dish is left out for a long period of time, the unit may revert back to a slower period of sampling.
In the embodiment, the reader uses three wavelengths in the optical measurement (more than three could also be used). Two of these wavelengths are used to determine the pH from the ratio of acid and base form concentrations of the indicator. This is determined by using the absorption coefficients of the acid and base forms of the indicator, and solving simultaneous equations for the absorption at the two wavelengths. Using a ratio makes the measurement relatively independent of the actual amount of the indicator added to the cuvette. Since the apparatus is to be as low cost as possible, it is another aspect of the reference example to incorporate a method of auto zeroing. In optical measurements, usually, a zero level measurement is performed with a sample blank prior to measuring the sample. The absorption levels of the blank are then subtracted from the sample reading to provide the net absorbance of the sample. In the apparatus, the third wavelength is chosen such that it shows very little absorption by the indicator, and is used as means of tracking changes in the zero level. Changes in the absorption level of this wavelength channel are then indicative of changes in the zero level, and the other two wavelengths which are used in the measurement can be zero corrected based on the changes measured at this third wavelength. This will correct for variations arising due to offsets, for example arising from different wall thickness cuvettes or coatings depositing out of solution onto the cuvette walls.
Another factor which affects the zero level is the temperature of the LEDs. Experiments have shown that the intensities of the three wavelengths vary with temperature, but not by the same absolute amount. A simple factory calibration of the apparatus provides coefficients for the relationship between the different wavelength LEDs. Any shift in the absorbance level of the third wavelength is due to effects of offsets (as described above) and temperature drift. The measured temperature can be used to calculate the thermal drift component, and the remainder of any change in the zero level of the third wavelength will be due to offset effects. The offset and temperature drift corrections can then be determined and applied to the other two wavelengths used in determining the pH.
FIG. 10 shows one embodiment the of reader unit according to the reference example. The reader unit 220 has a reader body 221 and a gripper 222 for receiving and retaining a culture vessel 224. The gripper may be of any convenient size to grip and carry a culture vessel. For example, the gripper is made of silicone elastomer, and sized to grip and retain a 35-mm culture dish. This enables a culture dish to be transported with the reader unit to enable monitoring to be continued outside the incubator.
The reader body 221 includes a recess 226 for a cuvette 228 to carry a sample of the fluid which is the same as that in the culture dish as discussed above.
Within the reader body, as shown in FIG. 11, there is an LED light source arrangement 230 comprised of three or more LEDs of different frequencies as discussed above directed to a light guide 232 so that the light beam passes across the slot 226 to an LED receiver assembly 236. The LED receiver assembly 236 includes receivers for each of the frequencies of the LED light source arrangement. Electronic circuitry 238 processes the various readings, and a battery 239 (underneath the electronic circuitry and shown by the broken line) makes the reader unit self-contained. Adjacent to the light source 230 is a second LED receiver 240 which measures and compensates for drift in the transmitting LED assembly 230 emitting light. An aerial 242 associated with the electronic circuitry transmits readings to a data logger within the incubator or to a monitoring device outside the incubator. The reader unit also includes a thermocouple 244 for measuring the temperature, and the electronic circuitry 238 can transmit temperature data as well as pH data.
A version of the reader unit as shown in FIGS. 10 and 11 may be supplied without the gripper. Such an apparatus can be used to monitor a whole incubator chamber and act as a warning device, setting off an alarm when the pH or the temperature moves outside preset limits.
As described above, in the related-art optical pH measurement, the zero level measurement is performed with a sample blank prior to measuring the sample, and the absorption levels of the blank are then subtracted from the sample reading to provide the net absorbance of the sample. The vicinity of the wavelength (700 nm) which shows very little absorption by the indicator is chosen as the third wavelength, and is used as means for tracking changes in the zero level. Changes in the absorption level of this wavelength channel are indicative of changes in the zero level. Therefore, the other two wavelengths which are used in the measurement must be zero corrected based on the changes measured at this third wavelength.